In Vitro Culture of Ovules and Embryos from Seedless Grapes (Vitis vinifera L.)

In Vitro Culture of vules and Embryos from Seedless Grapes (Vitis vinifera L.) P. Burger and P.. Goussard" 1) Nietvoorbij Researh Institute for Vitiulture and enology, Agriultural Researh Counil, Private Bag X026, 799 Stellenbosh, South Afria 2) Department of Vitiulture and enology, University of Stellenbosh, 7600 Stellenbosh, South Afria Submitted for publiation: May 1996 Aepted for publiation: tober 1996 Key words: Vitis vinifera, in vitro embryo ulture, ovule size, ovule age, plant growth regulators vules of stenospermoarpi seedless grapes were ultured under different onditions. The number of embryos was not signifiantly inreased when ovules of Musat Seedless were ultured on a medium supplemented with the plant growth regulators indole-- aeti aid and gibberelli aid ompared to the basal medium. No orrelation was found between the ovule size of Sultanina and the presene of embryos. Comparle perentages of embryos were found in small, medium and large ovules following weekly ulture from four to weeks after bloom. Varying results were obtained when ovules of four open-pollinated seedless ultivars were ultured on two basal media with or without ativated haroal. In all four ultivars the highest number of embryos was found at the later ulture date. With three ultivars the best results were obtained when ovules were ultured on the medium of Bouquet & Davis (1989), supplemented with ativated haroal. Isolated embryos of Musat Seedless and Sunred Seedless were ultured under various onditions and it was found that the sene of growth regulators resulted in the highest number of plantlets with normal leaves. Breeding new seedless tle grape ultivars has a high priority world-wide due to onsumer preferene. Conventional breeding tehniques limit the use of seedless ultivars to pollen parent only, resulting in a low perentage of seedless progeny. Development of embryo ulture tehniques, however, makes it possible to generate plants from the embryos of seedless plants and thus from seedless x seedless rosses. A muh higher frequeny of seedlessness is found in the progeny of suh rosses (Spiegel-Roy, Sahar & Baron, 1990). In stenospermoarpi ultivars a high number of the ovules possess normal embryo sas, fertilisation ours and embryos may develop (Pearson, 192; Barrit, 1970). Embryo development, however, is often arrested and normal seeds do not develop. By applying in vitro tehniques these embryos an be resued and allowed to develop into plants. Embryo ulture has been suessfully applied by a number of researhers, using various media and tehniques (either liquid or solid, supplemented with or without plant growth regulators) (Cain, Emershad & Tarailo, 198; Emershad & Ramming, 1984; Gray, Fisher & Mortensen, 1987; Barlass, Ramming & Davis, 1988). Embryos of stenospermoarpi grapes are le to germinate diretly from the ovule when ultured on an artifiial medium (Spiegel-Roy et al., 198). However, a ertain perentage of embryos fail to germinate while embedded in the ovule. To ensure the highest frequeny of germination, a number of authors found it neessary to exise embryos after a period in ulture (Cain et al., 198; Emershad & Ramming, 1984; Gray et al., 1987; Barlass et al., 1988). Various tehniques are applied to stimulate the growth of isolated embryos and their development into plants. Embryos obtained from ultured ovules appeared to be dormant, but old stratifiation of the isolated embryos at 4 C for six weeks failed to promote germination, while the addition of N 6 -Benzyladenine to the medium stimulated rapid germination (Gray et al., 1990). Bouquet & Davis (1989), however, found that BA was detrimental to plant development and that gibberelli aid was ineffetive in breaking dormany. The aim of this study was to investigate different fators whih may affet the suess of in vitro ulture of grape embryos.the effet of plant growth regulators, developmental stage and ovule size on embryo growth was investigated. The effet of different ulture onditions on the growth of isolated embryos was also studied. MATERIALS AND METHDS vule ulture: A standard proedure as desribed by Spiegel-Roy et al., (198) with appropriate modifiations was followed. Berries were olleted in the vineyard at different stages after pollination, rinsed under running tap water (20 min), surfae-sterilised for 1 min in 1% NaCl and rinsed twie in sterile water. vules were aseptially removed and ultured on the basal medium of Nitsh & Nitsh (1969) (), modified by inreasing the FeS 4.7H, onentration X, or on the basal medium of Bouquet & Davis (1989) (). Sigma agar was used. vules were ultured in petri dishes (90 mm x mm) under standard onditions of 26 ± 2 C and 16 h ool-white fluoresent (G.E.C. 6 W) illumination. In some of the experiments (see below) -indoleaeti aid (IAA), gibberelli aid (GA ), N -Benzyladenine (BA) and indole- butyri aid (IBA) were added to the media. Media were sterilised at 121 C for 1 min and the plant growth regulator solutions were filter-sterilised (0,2 um Millipore filters) Aknowledgements: The tehnial assistane of Mrs E. Visagie and Miss A.E. Theron is muh appreiated. The authors sinerely wish to thank Prof. R. de V. Pienaar and Dr I.A. Trautmannfor reading the manusript ritially and the Tle Grape Industry for finanial support. 1

2 In Vitro Culture of vules and Embryos and added after autolaving. The effet of GA and IAA: Musat Seedless ovules were ultured on or supplemented with 1x M IAA and 1x M GA. vules were olleted at five, seven, nine and eleven weeks after bloom and ultured as desribed ove. After 12 ± 1 weeks in ulture the perentage of germinated ovules was determined for eah treatment. The Spiegel-Roy et al., (198) tehnique was adapted by exising the remaining (ungerminated) ovules and ulturing the isolated embryos on in darkness at 26 ± 2 C. Germinated embryos were transferred to a growth hamber and ultured under standard onditions. The resulting plants were miropropagated and alimatised in a greenhouse. The effet of ultivar, basal medium and addition of ativated haroal: vules of open-pollinated Musat Seedless, Flame Seedless, Festival Seedless and a seletion, 72-41, were ultured at six and eight weeks after bloom. Mean ovule masses at six weeks after bloom were 6,2 mg;, mg; 6,22 mg and,84 mg respetively, while it was.6 mg; 4,48 mg; 7,2 mg and 6,6 mg at eight weeks after bloom. Two basal media ( and ) with and without ativated haroal (2 g/l) were inluded in the experiment. The standard proedure desribed ove was followed and isolated embryos were ultured on. The effet of ovule size: Sultanina ovules were olleted weekly from four to weeks after bloom and visually grouped into three lasses aording to size (small, medium and large). Size riteria varied aording to ulture date (weeks after bloom). ne hundred ovules of eah size lass were ultured on for 12 ± 1 weeks after whih their embryos were exised. Isolated embryos were ultured on. All the experiments desribed ove were arranged in a fatorial design and repliated in either two or three bloks. An experimental unit onsisted of five petri dishes with ovules per petri dish. The number of embryos germinating from intat ovules was ounted and afterwards the ungerminated ovules were disseted to determine the number ontaining embryos. The total embryo perentage ould thus be alulated. The number of ovules per unit differed (attributed to ontamination) and a working logit transformation was thus applied. The transformed values were then analysed. In order to ompare treatment means Student's t- LSD test was applied at % signifiane. Treatments were developmental stage (i.e. time measured as weeks after bloom), different media (inluding the addition of plant growth regulators and ativated haroal) and ovule size (small, medium and large). Development of exised embryos: During 1991 isolated embryos of Musat Seedless were submitted to different treatments to stimulate growth and further development into normal seedlings. Treatments were as follows: (1) + 16h illumination at 26 ± 2 C (ontrol), (2) + darkness at 4 C, () + darkness at 26 ± 2 C; (4) + BA + darkness at 26 ± 2 C, () + GA + IAA + darkness at 26 ± 2 C, (6) + GA + darkness at 26 ± 2 C. Based on the results of the previous year, the following media were inluded in a similar experiment with Sunred Seedless and isolated embryos were submitted to the following treatments (1992): (1) + darkness at 26 ± 2 C (ontrol), (2) + darkness at 26 ± 2 C, () + BA + darkness at 26 ± 2 C, (4) + GA +IAA + darkness at 26 ± 2 G,() + GA + darkness at 26 ± 2 C. Plant growth regulator onentrations were 1 x M IAA, 1 x " 6 M GA and 1 x ' 6 M BA, respetively. Treatments inluded the addition of plant growth regulators to the medium and different illumination periods and were repliated in three bloks during 1991. An experimental unit onsisted of five petri dishes with five embryos per petri dish (2 embryos per blok). During 1992 treatments were repliated in five bloks and an experimental unit onsisted of six petri dishes with embryos per dish ( embryos per blok). After three weeks, when petri dishes were transferred to standard illumination and temperature onditions, observations were made by ounting the number of germinated embryos. Data on the number of embryos whih eventually developed into plantlets with normal leaves were reorded two months later. Data were subjeted to a working logit transformation and analysis of variane was performed as desribed ove. Miropropagation and alimatisation: Although normal healthy plants developed from the ultured embryos, normal growth of the otyledons was frequently observed. When morphologially normal stems and leaves developed from embryos with normal otyledons, these shoots were removed and rooted on medium ontaining 6,2 x M IB A. In all experiments, when leaves developed, plantlets were transferred from petri dishes to in 0ml glass jars. Plantlets were miropropagated by onenode mirouttings on medium supplemented with 4 x M BA to enburage shoot proliferation. Resulting shoots were rooted on medium supplemented with IBA. Rooted plants were removed from the jars, the roots washed under running water, dipped in a 1% Benlate solution and planted in an unsterilised mixture of ommerial "speeding" mix (Kompel) and vermiulite (1:1) in polystyrene seedling trays. Eah tray was overed with a plasti bag and transferred to a greenhouse at 26 ± 2 C and 16 h illumination. After days the plasti was gradually ruptured., followed by omplete removal after 1 days. RESULTS AND DISCUSSIN Polyembryos and seondary embryos obtained from one ovule were regarded as one embryo to interpret results. The effet of GA and IAA: nly a very small number of embryos were le to germinate from intat Musat Seedless ovules and for eah ulture date no signifiant differenes were found for ovules ultured on media with and

In Vitro Culture of vules and Embryos in a X + IAA + GA, I 4 U_ Z 2 1 7 9 11 WEEKS AFTER BLM FIGURE l(a) The effet of plant growth regulators on the perentage of embryos germinating from intat Musat Seedless ovules (: Basal medium of Nitsh & Nitsh; GA : Gibberelli aid; IAA: -Indoleaeti aid). Perentages identified by the same symbols do not differ signifiantly (p<0,0). 40 CD 20 a + IAA + GA I CC Q. 7 9 WEEKS AFTER BLM FIGURE l(b) The effet of plant growth regulators on the perentage of embryos germinating from intat Musat Seedless ovules as perentage of the total number of embryos (: Basal medium of Nitsh & Nitsh; GA : Gibberelli aid; IAA: -Indoleaeti aid). Perentages identified by the same symbols do not differ signifiantly (p<0,0). 2 11 C/ 20 C m gxj + IAA + GA 1 0 < d d I 7 9 WEEKS AFTER BLM FIGURE l() The effet of plant growth regulators on the total perentage of embryos obtained from intat as well as disseted Musat Seedless ovules (: Basal medium of Nitsh & Nitsh; GA_,: Gibberelli aid; IAA: -Indoleaeti aid). Perentages identified by the same symbols do not differ signifiantly (p<0,0). 11

4 In Vitro Culture of vules and Embryos Musat Seedless Festival Seedless C p > " CC m S 20 <? 1 + AC + AC C 2 CQ 20 1 + AC + AC CC Q. Q_ de d de de 6 weeks 8 weeks WEEKS AFTER BLM 6 weeks ~ 8 weeks WEEKS AFTER BLM a) b) C 0 2 CC CD go )ENTAGE CJl CC Q. 0 " n - - /// + AC + AC Flame Seedless 72-41 P -j 6 weeks 8 weeks WEEKS AFTER BLM b a g \$ /IBRYS N> n 20 I Q_ C) d) 0 ^ rn ^ + AC ^ + AC _ * - a XV\ _ / /, \ \ ' ^ //, l\* be *y\ / / s\\ ~^~7- T-r-A KV / // ' XX^^? 6 weeks 8 weeks WEEKS AFTER BLM FIGURE 2 The effet of ultivar, medium and developmental stage of ovules on the perentage of embryos obtained (: Basal medium of Bouquet & Davis; : Basal medium of Nitsh & Nitsh; AC Ativated haroal). Perentages identified by the same symbols do not differ signifiantly (p<0,0).

In Vitro Culture of vules and Embryos 2 20 <r C in u_ J CD J EC D. 1 de d ef de bed h ; h efg fg def 6 7 8 WEEKS AFTER BLM FIGURE The effet of ovule size on the perentage of Sultanina embryos obtained. vules were ultured on (: Basal medium of Nitsh & Nitsh). Perentages identified by the same symbols do not differ signifiantly (p<0,0). without plant growth regulators (Fig. la). Preliminary results were Sultanina ovules ultured with the inlusion of different onentrations and ombinations of BA, GA and IAA indiated that these growth regulators were not essential for embryo reovery (Burger, 1992). When onsidering the number of embryos that germinated from intat ovules as perentage of the total number of embryos, a higher number germinated when ultured on medium supplemented with growth regulators. These differenes, however, were not signifiant (Fig. Ib). However, despite the indiation of plant growth regulators promoting germination from intat ovules, it was indiated that a higher total number of embryos (as perentage of ovules) is found when ovules are ultured on medium without plant growth regulators (Fig. I). For eah ulture date signifiant differenes in the number of embryos were found only for the ovules ultured nine weeks after bloom, in whih ase the higher embryo perentage was found in the ovules ultured on the basal medium (Fig. I). In order to obtain the most plants, the ovules need to be disseted and the isolated embryos ultured. This observation onfirms that of a number of authors who found it neessary to disset the embryos from the ovules after a ertain ulture period (Barlass el al., 1988; Cain et al, 198; Gray et al, 1987; Emershad et al, 1989; Gray et al, 1990; Ramming et al, 1990). The effet of ultivar, basal medium and addition of ativated haroal: Signifiantly more embryos were obtained with Musat Seedless ovules when ultured eight weeks after bloom on medium supplemented with ativated haroal irrespetive of basal medium (Fig. 2a). The same trend was observed for Festival Seedless (Fig. 2b). However, in this instane the results for the two basal media supplemented with ativated haroal differed signifiantly. The highest number of embryos was obtained from ovules ultured on (Fig. 2b). For both Flame Seedless and 72-41 the highest numbers of embryos were obtained from ovules ultured on media supplemented with ativated haroal (Fig. 2,2d). It seems that ativated haroal has no benefiial effet at six weeks after bloom. These results are in aordane with those of Bouquet & Davis (1989), who also found that the addition of ativated haroal inreased the perentage of embryos, irrespetive of ulture date. Although not statistially onfirmed, it was observed that the ultivars with the highest embryo perentages had ovules with higher mean masses. Cain et al, (198), Goldy & Amborn (1987) and Spiegel-Roy et al, (1990) related differenes in embryo perentages to the ovule size of different ultivars. More embryos were obtained from ultivars with large ovules than from those with small ovules. The highest number of embryos for all four ultivars was obtained at eight weeks after bloom. The effet of ovule size: No orrelation was found between ovule size and the presene of embryos in Sultanina ovules. Contrary to expetation, a relatively high perentage of small ovules yielded embryos and the highest perentage of ovules that ontained embryos at seven and eight weeks after bloom was lassed as small ovules (Fig. ). S. Afr. J. Enol. Vide., Vol. 17, No. 2,1996

6 In Vitro Culture of vules and Embryos TABLE 1 The effet of different treatments on embryo growth and plant development in vitro (Musat Seedless, 1991). y) Treatment Number of embryos Number of germinating embryos p<0,0 Number of plants with normal leaves p<0,0 1 64 * (4,7%) ' 2 62 9 b 2(7,1%)"" 6 4 b 26 (40,0%) * 4 6 * 19 (29,2%) b 68 46 b 29 (42,6%) a " 6 64 a (1,6%)" b y) Treatments applied to isolated embryos: (1) + 16 h illumination at 26 ± 2 C (Control)., (2) + darkness at 4 C., () + darkness at 26 ± 2 C, (4) + BA + darkness at 26 ± 2 C, () + GA + IAA + darkness at 26 ± 2 C., (6) + GA + darkness at 26 ± 2 C. Embryos ultured in darkness were transferred to onditions of 16 h illumination at 26 ± 2 C after weeks. Germinating embryos were ounted at this stage and the data for plants with normal leaves were reorded two months afterwards. Values in olumns designated by the same symbol do not differ sigfniflantly. Values in brakets represent the perentage of embryos that developed into plants. TABLE 2 The effet of different treatments on embryo growth and plant development in vitro (Sunred Seedless, 1992). Medium Number of embryos Number of germinating embryos Number of plants with normal leaves 140 7 a 8 (9,%) * 12 98 80 (60,7%) ' + BA 140 92 b 9(42,1%)" + GA 18 86 C 1 (6,9%) ' + GA + IAA 12 8 b 20 (1,0%) " Embryos were ultured in darkness for weeks, before transferene to 16 h illumination at 26 ± 2 C Germinating embryos were ounted at this stage and the data for plants with normal leaves were reorded two months afterwards. Values between brakets showt the perentage of embryos whih developed to plants with normal leaves.. Values in olumns designated by the same symbol do not differ sigfniflantly (p<0,0).

In Vitro Culture of vules and Embryos 7 These results are in ontrast to those of Bouquet & Davis (1989), who observed a orrelation between the size of the ovules and vile embryos within a given ultivar. Embryo perentages in small ovules dereased when the time span inreased, while the highest perentage of medium and large ovules ontaining embryos was found at nine weeks after bloom. A gradual inrease in perentage of embryos was found in ovules that were ultured at later developmental stages (up to nine weeks) than at four and five weeks after bloom and it was onluded that Sultanina ovules should be ultured at nine weeks after bloom and only medium and large ovules should be ultured (Fig. ). Development of exised embryos: The addition of BA stimulated further development of embryos and germination, but not the development of plants with morphologially normal leaves. The number of embryos (exised from Musat Seedless ovules) that eventually produed leaves were signifiantly higher when embryos wer subjeted to a 16 h illumination period (ontrol), ompared to those obtained when embryos were ultured on medium ontaining BA (Tle 1). During*the following year the highest perentage of plants with normal leaves was obtained when Sunred Seedless embryos were ultured on the two basal media. The results for the two basal media did not differ signifiantly, but the number of normal plants was signifiantly higher ompared to the treatments with plant growth regulators inluded in the media (Tle 2). Gray et al., (1990) found that the addition of 1 x " M BA to solid Murashige & Skoog (MS) medium stimulated rapid growth of exised embryos. However, normalities were reorded in these embryos. In the present study it was found that although BA stimulated embryo germination, embryos and plantlets developed normalities, as was also reported by Bouquet & Davis (1989). The results of the present study regarding the ineffiieny of GA to break dormany are in aordane with those of Emershad & Ramming (1984) and Bouquet & Davis (1989). CNCSINS obtained from seedless x seeedless rosses fruited during 1994. LITERATURE CITED BARLASS, M., RAMMING, D.W. & DAVIS, H.P., 1988. In-ovulo embryo ulture: a breeding tehnique to resue seedless x seedless tle grape rosses. Aust. Grapegrower & Winemaker 292, 12-12. BARRITT, B.H., 1970. vule development in seeded and seedless grapes. Vitis 9,7-14. BUQUET, A. & DAVIS, H.P., 1989. Culture in vitro d'embryons de vigne (Vitis vinifera L.) appliquee a la seletion de varietes de raisins de tle sans pepins. Agronomi 9, 6-74. BURGER, P., 1992. Embryo ulture and its appliation in tle grape breeding. M.S. Agri. thesis. University of Stellenbosh, Private Bag XI, 7602 Matieland, Stellenbosh. CAIN, D.W., EMERSHAD, R.L. & TARAIL, R.E., 198. In-ovulo embryo ulture and seedling development of seeded and seedless grapes (Vitis vinifera L.) Vitis 22, 9-14. EMERSHAD, R. L. & RAMMING, D.W., 1984. In-ovulo embryo ulture of Vitis vinifera L. v. "Thompson Seedless". Amer. J. Bot. 71, 87-877. EMERSHAD, R. L. & RAMMING, D.W. & SERPE, M.D., 1989. In-ovulo embryo development and plant formation from stenospermi genotypes of Vitis vinifera L. Amer, J. Bot. 76, 97-402. GLDY, R.G., & AMBRN, U., 1987. In vitro ulturility of ovules from seedless grape lones. HortSiene 22, 92. GRAY, D.J., FISHER, L.C. & MRTENSEN, J.A., 1987. Comparison of methodologies for in-ovulo embryo resue of seedless grapes. HortSiene 22, 14-1. GRAY, D.J., MRTENSEN, J.A., BENTN, C.M., DURHAM, R.E. & MRE, G.A., 1990. vule ulture to obtain progeny from hybrid seedless bunh grapes. J. Amer. So. Hort. Si. 11, 19-24. NITSCH, J.P. & NITSCH, C, 1969. Haploid plants from pollen grains. Siene 16, 8-87. PEARSN, H.M., 192. Parthenoarpy and seed ortion in Vitis vinifera. Pro. Amer. So. Hort. Si. 29, 769-17. RAMMING, D.W., EMERSHAD, R.L., SP1EGEL-RY, P., SAHAR, N. & BARN. I., 1990. Embryo ulture of early ripening seeded grape (Vitis vinifera) genotypes. HortSiene 2, 9-42. SPIEGEL-RY, P., SAHAR, N., BARN, J. & LAVI, U., 198. In vitro ulture and plant formation from grape ultivars with ortive ovules and seeds. J. Amer. So. Hort. Si.. 1, 9-112. SPIEGEL-RY, P., SAHAR, N., BARN, I., 1990. Seedless x seedless grape progeny: Tehnique, results and perspetive. In: Pro. th Intern. Symp. Grape Breeding, St. Martin/Pfalz, FR of Germany, 12-16 September 1989. pp. 42-48. Plants were obtained by ulturing the ovules and embryos of open-pollinated seedless ultivars and it was lear that a number of fators ontributed to the number of plants that eventually developed. Cultivar and ovule age at ulturing greatly influened the number of embryos resued and it was lear that ovules should not be ultured earlier than six weeks after bloom. Depending on ulture date, the addition of ativated haroal to the medium resulted in higher numbers of embryos. It may be onluded that illumination has no effet in the initial development of isolated embryos and that these embryos should be ultured on medium without growth regulators. The pratie of ulturing intat ovules, followed by exision of the ovules and ulturing of isolated embryos on medium without plant growth regulators, ensures the preservation of the widest spetrum of geneti diversity from a speifi ross. This proedure is now routinely followed in the tle grape breeding programme of Nietvoorbij and the first plants